The traditional “cascade” view of blood coagulation is illustrated in FIG. 1 and consists of two converging pathways: the intrinsic or “contact” pathway was originally defined based on adding clay or other “contact reagents” to blood in a test-tube, and the extrinsic pathway that is now believed to be the pathway by which clotting is initiated following tissue injury. Both of these pathways culminate in the production of blood coagulation factor Xa (FXa, a serine protease) by pathways that depend on membranes. In one of these, factor IXa (FIXa, also a serine protease) and its cofactor factor VIIIa (FVIIIa) activate factor X (FX) in a reaction that is dependent on activated platelet membranes (intrinsic pathway). In the other (extrinsic pathway), FX is activated by another plasma serine protease factor VIIa (FVIIa) in conjunction with a cell-membrane located (thus, “extrinsic” to the plasma) cofactor, tissue factor (TF). These two pathways join at the “common pathway,” in which FXa along with its cofactor factor Va (FVa), activates prothrombin to thrombin in the final step in the blood coagulation cascade. While it is generally believed that the exposure of TF to plasma following tissue damage is the trigger that initiates blood coagulation, it is also clear that the platelet-membrane-dependent complexes (Xa-Va, IXa-VIIIa) are essential to amplify the process so that a clot will form. Indeed, most of the familial coagulation disorders involve defects in these four proteins.
Prothrombin activation is accomplished by the FXa-FVa enzyme complex, called “prothrombinase” in the presence of Ca2+, and negatively charged membranes (Mann, et al. (1990) Blood 76(1):1-16; Rosing, et al. (1980) J. Biol. Chem. 255(1):274-83). In vivo, the membranes derive from activated platelets in the form of vesicles (Sandberg, et al. (1985) Thromb. Res. 39(1):63-79; Sims, et al. (1989) J. Biol. Chem. 264(29):17049-57) upon whose surface phosphatidylserine (PS) is exposed (Comfurius, et al. (1990) Biochim. Biophys. Acta 1026(2):153-60), having been buried on the cytoplasmic surface of resting platelets (Schick, et al. (1976) J. Clin. Invest. 57(5):1221-6). PS is known to have a specific role in prothrombin activation (Jones et al. (1985) Thrombosis Res. 39, 711-724; Comfurius et al. (1994) Biochemistry 33 10319-10324), but the nature of that role has not previously been understood. Because two bonds in prothrombin must be cut, activation can proceed via two possible proteolytic intermediates (FIG. 4), meizothrombin (MzIIa), probably the major intermediate in vivo (Rosing and Tans (1988) Thromb. Haemost. 60(3):355-60; Nesheim and Mann (1983) J. Biol. Chem. 258(9):5386-91) and prethromin 2 plus fragment 1.2 (Pre2 & F1.2; FIG. 2) (Nesheim and Mann (1983) supra; Krishnaswamy, et al. (1987) J. Biol. Chem. 262(7):3291-9). Both FVa and PS-membranes are thought to direct activation through the meizothrombin intermediate, but PS has the major role in this regard (Boskovic, et al. (2001) J. Biol. Chem. 276(31):28686-93; Banerjee, et al. (2002) Biochemistry 41 (3):950-7; Wu, et al. (2002) Biochemistry 41(3):935-49). The fact that PS has this significant role and that it also alters the activity of factor Xa (Koppaka et al. (1996) Biochemistry 35:7482) implies a regulatory role for this platelet lipid.
Coagulation factor IX also plays a pivotal role in blood coagulation as shown by the bleeding tendency associated with congenital factor IX deficiency (hemophilia B). The activated form of X, FIXa, plays a key role in thrombin generation at the platelet plug by binding with FVIIIa on platelet membranes to form the Xase complex that activates X to Xa. Negatively charged phospholipids, especially PS, are also critical to this process. PS-containing membranes increase the kcat of the factor VIIIa-IXa complex by more than a 1000-fold (Gilbert et al. (1996) J. Biol. Chem. 271 11120). FVIIIa and FIXa bind specifically and with high affinity to PS-containing membranes. However, as for the prothrombinase complex, the exact role of PS in the activation of FX is not known.
Synthetic membrane preparations, which can be derived from phospholipid extracts of platelets, mammalian brain or lung, or soybeans, are commonly used in current clotting assays. More recently, synthetic membranes prepared from mixtures of purified synthetic lipids have been used to improve reproducibility and shelf life of assays. Synthetic phospholipids are also commercially available (e.g., from Avanti® Polar Lipids Inc; Alabaster, Ala.). Even these synthetic membrane preparations suffer from the drawbacks that (1) they are labor-intensive to produce, (2) each batch must be individually calibrated to match clotting times to existing standards (see, e.g., U.S. Pat. No. 6,596,543 B2 to Wang et al.), and (3) they still have only a limited shelf-life.
Accordingly, there is a need in the art for improved reagents and methods for performing clotting assays and other assays of clotting factor activity.